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Chromatography:

Definition:

Definition Ion-exchange chromatography (or ion chromatography ) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. 2

Ion-exchange chromatography:

Ion-exchange chromatography The solution to be injected is usually called a sample , and the individually separated components are called analytes It can be used for almost any kind of charged molecule including large proteins , small nucleotides and amino acids. It is often used in protein purification , water analysis. 3

Anion exchange chromatography:

Procedure:

Procedure A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. The mobile phase (buffered aqueous solution) carries the sample from the loop onto a column that contains some form of stationary phase material. Stationary phase material is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. 11

Procedure:

Procedure The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions. 12

Procedure:

Procedure The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance. A chromatography data system (CDS) is usually needed to control an IC. 13

Procedure:

Procedure Dr Gihan Gawish 14

Separating proteins :

Separating proteins Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase. 15

Affect of pH in the separation of proteins:

Affect of pH in the separation of proteins By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively-charged beads, whereas a negatively charged protein would not. 16

Applications….:

Used for::

Used for: Antibody Purification Monoclonal antibodies produced in mouse ascites can be separated from other components of the ascites fluid. Immunoglobulins can be separated from albumin, transferrins and proteases. 21